1. Field of the Invention
The present invention relates to a method for producing useful metabolites, particularly those derived from acetyl-coenzyme A (acetyl-CoA). The present invention also relates to novel bacteria useful in the production method.
2. Brief Description of the Related Art
Conventionally, useful metabolites such as L-amino acids, their intermediates, and other chemicals of bacterial metabolism are produced by methods in which bacterial strains isolated from natural sources, or mutants thereof, have been modified to enhance their productivity.
Sugar is the main source of carbon in a microorganism which is suitable for fermentation. The Embden-Meyerhof and pentose phosphate (pentose-P) pathways are the two preliminary routes of intermediary sugar metabolism in a microorganism. A third route, the Entner-Doudoroff pathway, is also known, as are some of the connections with carboxylic acid pathways. During glycolysis, glucose is metabolized to main intermediate compounds, such as phosphoenolpyruvate, pyruvate, and acetyl-coenzyme A, which are used as constituents in the formation of many cellular compounds, such as L-amino acids, purines and pyrimidines, vitamins etc. Also, generation of energy (ATP and NADH) occurs during glycolysis. Pyruvate formed after glycolysis is often converted back to phosphoenolpyruvate (PEP) by phosphoenolpyruvate synthase encoded by the pps gene, or to acetyl-CoA by pyruvate dehydrogenase encoded by the pdh gene etc. One of the above-mentioned compounds, acetyl-CoA, is formed from pyruvate via pyruvate dehydrogenase, and accompanied by the release of CO2. This loss of one carbon atom results in decreased production yields of useful compounds derived from acetyl-CoA. Two enzymes of the bifidum pathway, D-xylulose-5-phosphate phosphoketolase (also known as “phosphoketolase”) and fructose-6-phopshate phosphoketolase, have been reported. D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9) catalyzes the phosphate-consuming conversion of xylulose-5-phosphate to glyceraldehyde-3-phosphate and acetylphosphate, with the concommitant release of one molecule of water. Fructose-6-phopshate phosphoketolase (EC 4.1.2.22) catalyzes the phosphate-consuming conversion of fructose-6-phosphate to erythrose-4-phosphate and acetylphosphate, with the concommitant release of one molecule of water. Both enzymes form acetylphosphate, the precursor of acetyl-CoA, without losing carbon via CO2. D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9) has been reported in bacteria belonging to the genera Acetobacter (Schramm, M. et al, J. Biol. Chem., 233(6), 1283-8 (1958)), Bifidobacterium (Sgorbati, B. et al, Antonie Van Leeuwenhoek. 42(1-2), 49-57 (1976); Grill, J. P. et al Curr Microbiol., 31(1), 49-54 (1995)), Lactobacillus (Posthuma, C. C. et al, Appl. Environ. Microbiol., 68(2), 831-7 (2002)), Thiobacillus (Greenley, D. E. and Smith, D. W., Arch. Microbiol., 122, 257-261 (1979)), in yeasts belonging to the genera Candida, Rhodotorula, Rhodosporidium, Pichia, Yarrowia, Hansenula, Hansenula, Kluyveromyces, Saccharomyces, Trichosporon, Wingea (Evans, C. T. and Ratledge, C., Arch. Microbiol., 139, 48-52 (1984); Ratledge, C. and Holdsworth, J. E., Appl. Microbiol. Biotechnol., 22, 217-221 (1985)). Fructose-6-phopshate phosphoketolase (EC 4.1.2.22) has been reported in bacteria, such as Acetobacter xylinum (Schramm, M. et al, J. Biol. Chem., 233(6), 1283-8 (1958)), Bifidobacterium globosum and Bifidobacterium dentium (Sgorbath, B. et al, Antonie Leeuwenhoek, 42, 49-57 (1976)), Bifidobacterium bifidum, Gardnerella vaginalis (Gavini, F. et al, Anaerobe, 2, 191-193 (1996)), and yeasts, such as Rhodotorula graminis, Rhodotorula glutinis, Candida sp., Candida tropicalis, Saccharomyces pastorianus (Whitworth, D. A. and Ratledge, C., J. Gen. Microbiol., 102, 397-401 (1977)). It has been reported that in some organisms both activities are represented by one enzyme (see, for example, the articles of Schramm, M. et al (J. Biol. Chem., 233(6), 1283-8 (1958)); Sgorbati, B. et al (Antonie Van Leeuwenhoek. 42(1-2), 49-57 (1976)); Meile, L. et al (J. Bacteriol., 183(9), 2929-36 (2001))). Phosphoketolase genes from two species have been cloned and their sequences determined. These are the xfp gene which encodes D-xylulose-5-phosphate phosphoketolase/fructose-6-phopshate phosphoketolase from Bifidobacterium lactis (Meile, L. et al, J. Bacteriol., 183(9), 2929-36 (2001)), and the xpkA gene which encodes D-xylulose-5-phosphate phosphoketolase from Lactobacillus pentosus (Posthuma, C. C. et al, Appl. Environ. Microbiol., 68(2), 831-7 (2002)). A seach of the Microbial Genome database provided by the National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=&DB=genome) revealed several genes encoding for putative phosphoketolases.
Methods for improving the ability of yeast to produce ethanol from xylose by introducing genes for xylose reductase, xylitol dehydrogenase, and additionally phosphoketolase are known (WO2003078643). However, effects of using the phosphoketolase gene for the elimination of carbon dioxide have never been reported.